Molecular cloning and expression of the functional rat C5a receptor.
The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.
Rothermel
E
E
Abteilung für Immunologie, Georg-August-Universität, Göttingen, Germany.
Zwirner
J
J
Vogt
T
T
Rabini
S
S
Götze
O
O
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002633:Chemotaxis; D003001:Cloning, Molecular; D015936:Complement C5a / Q000378:metabolism*; D004285:Dogs; D015870:Gene Expression; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D002118:Calcium